J. Agric. Food Chem. 2008, 56, 4577–4583
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Effects of Fly Attack (Bactrocera oleae) on the Phenolic Profile and Selected Chemical Parameters of Olive Oil ´ ANA MARIA GOMEZ -CARAVACA,† LORENZO CERRETANI,*,‡ ALESSANDRA BENDINI,‡ ANTONIO SEGURA-CARRETERO,*,† ´ ´ ALBERTO FERNANDEZ -GUTIERREZ ,† MICHELE DEL CARLO,§ § DARIO COMPAGNONE, AND ANGELO CICHELLI| Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva s/n, E-18071 Granada, Spain, Department of Food Science, University of Bologna, Piazza Goidanich 60, I-47023 Cesena (FC), Italy, Department of food Science, University of Teramo, Via C. Lerici 1, I-64023 Mosciano Stazione (TE), Italy, and Department of Science, University of Pescara and Chieti, Viale Pindaro 42, I-65127 Pescara, Italy
The phenolic fraction of virgin olive oil influences both its quality and oxidative stability. One of the principal threats of the quality of olive fruit is the olive fly (Bactrocera oleae) as it alters the chemical composition. The attack of this olive pest has been studied in order to evaluate its influence on the quality of virgin olive oil (free acidity, peroxide value, fatty acid composition, water content, oxidative stability, phenols, and antioxidant power of phenolic fraction). The study was performed using several virgin olive oils obtained from olives with different degrees of fly infestation. They were acquired in different Italian industrial mills from the Abruzzo region. Qualitative and quantitative analyses of phenolic profiles were performed by capillary electrophoresis-diode array detection, and electrochemical evaluation of the antioxidant power of the phenolic fraction was also carried out. These analyses demonstrated that the degree of fly attack was positively correlated with free acidity (r ) 0.77, p < 0.05) and oxidized products (r ) 0.58, p < 0.05), and negatively related to the oxidative stability index (r ) -0.54, p < 0.05) and phenolic content (r ) -0.50, p < 0.05), mainly with secoiridoid compounds. However, it has been confirmed that the phenolic fraction of olive oil depends on several parameters and that a clear correlation does not exist between the percentages of fly attack and phenolic content. KEYWORDS: Virgin olive oil; phenols; qualitative parameters; oxidative stability; capillary electrophoresis; olive soundness
INTRODUCTION
Virgin olive oil is obtained from the fruit of the olive tree (Olea europaea L.) solely by mechanical or other physical means under conditions that do not alter its properties and must not undergo any treatments other than washing, decantation, centrifugation, or filtration (1). These processes maintain volatile and other minor compounds such as phenols that enhance the characteristic flavor of virgin olive oil (2). Stability is not a standard parameter used to measure quality. However, it provides information about the hypothetical shelf life of the oil. In particular, lower stability indicates a poorer * To whom correspondence should be addressed. Tel: +390547338121. Fax: +390547382348. E-mail:
[email protected]. † University of Granada. ‡ University of Bologna. § University of Teramo. | University of Pescara and Chieti.
quality (e.g., greater acidity, higher peroxide values and extinction coefficients, and lower sensorial score). It has been shown that 78% of the stability, evaluated by Rancimat, is due to the combined effect of two variables, namely, phenolic compounds and the oleic/linoleic (O/L) ratio. Phenolic compounds can be active as antioxidants and also can inhibit the free radical chain reaction (3). Their antioxidant properties and in particular their hydrogen-donating capacities are modulated by the presence of different chemical groups in the phenol backbones. Mainly, phenolic compounds having an o-catechol group in their structure such as those found in virgin olive oil (Figure 1), such as hydroxytyrosol and its oleosidic forms, are powerful antioxidants (4, 5). Using different assays, Carrasco-Pancorbo et al. (3) evaluated the antioxidant capacity of different phenolic compounds and concluded that among them, hydroxytyrosol, oleuropein, and decarboxy-methyl oleuropein aglycons with an o-catecholic structure exhibited the
10.1021/jf800118t CCC: $40.75 2008 American Chemical Society Published on Web 06/04/2008
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Go´mez-Caravaca et al.
Figure 1. Structures of the phenolic compounds under study.
strongest antioxidant activity. In contrast, monohydroxylated phenols as tyrosol and ligstroside aglycon had very poor radicalscavenging activity. Phenolic compounds have a positive effect on the health, sensory properties, and oxidative stability of olive oil (2, 6–9). But despite that it is well known that the phenolic fraction is influenced not only by the olive cv. but also by climatic and environmental conditions (10, 11), agronomic practices, and technological process (10–14). Plants are subject to attacks from different organisms and as a result have evolved a complex, integrated defense system against potential pathogenic organisms to ensure survival, growth, and development. It has been shown that plants respond to pathogenic attack by synthesizing compounds that activate the defense system in fruits (15). The quality of virgin olive oil is strongly related to the health status of the fruit from which it is extracted. One of the most detrimental enemies of the quality of olive oil is the olive fruit fly (Bactrocera oleae). This insect can reduce oil yield, affect quality parameters (acidity, peroxide value, ultraviolet (UV) absorbance, and organoleptic quality), and negatively alter the chemical composition (sterols, phenols, fatty acid, and volatile fraction) (12, 16–24). The severity of the negative effects depends on the stage of the development of the olive fly, the intensity of the attack, and olive variety. It has been shown that olive oils produced from fruits that have been attacked by the olive fly present an increase in acidity, peroxide values, and UV absorbance. Moreover, it has been demonstrated that the phenolic content and total amount of volatile compounds decrease, and no significant variations can be observed in fatty acid composition (24, 25). While there are several publications about the influence of Bactrocera oleae on the qualitative
parameters of olive oil, potential variations in the phenolic profile have not been considered in depth. Traditionally, free acidity and peroxide values have been considered the qualitative chemical parameters of virgin olive oil (1). Nevertheless, in recent years the evaluation of phenolic compounds as a qualitative parameter has been proposed (26). The first aim of this investigation was to assess different qualitative parameters of olive oil in various commercial olive oil samples depending on the percentage of fly attack. The second aim was related to the study of changes in the quality and oxidative stability of these olive oils, with particular emphasis to correlations with the phenolic profile and antioxidant power of the phenolic fraction. This statistical treatment of data allowed us to determine which qualitative parameter of olive oil was more robust and less influenced by all the variables. MATERIALS AND METHODS Reagents, Stock Solutions, and Reference Compounds. 3,4Dihydroxyphenylacetic acid (dopac) was obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA), and oleuropein (oleuropein glucoside) was obtained from Extrasynthe`se (Genay, France). The stock solutions of these two analytes were prepared in methanol/water (50/50, v/v) at a concentration of 500 µg/mL in the case of dopac and 6000 µg/mL for oleuropein glucoside. Dopac was used for the quantification of simple phenols present in the extracts of olive oil, and oleuropein glucoside was used to make the calibration curves for the quantification of lignans and complex phenols. Sodium hydroxide was purchased from Merck (Darmstadt, Germany); sodium tetraborate (borax) was obtained from Sigma and was used as running buffer at different concentrations and pH values. Double-deionized water with a conductivity less than 18.2 MΩ was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). Hydranal-Titran 2 and Hydranal-solvent oil (solvents used to measure
Olive Fruit Quality vs Virgin Olive Oil Characteristics
J. Agric. Food Chem., Vol. 56, No. 12, 2008
Table 1. Information Relative to Olives (Cultivars, Area of Production, Healthy State) and Corresponding Oil Samples (Code and Technological System of Their Production) code
olive varieties
town of production
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32
Dritta, Leccino Dritta, Intosso Intosso Leccino Dritta, Leccino Dritta Dritta Dritta Dritta Carpinetina Leccino Gentile Gentile Dritta, Leccino Tortiglione Mix Leccino Gentile Mix Mix Gentile Intosso Mix Dritta Leccino Mix Dritta Mix Mix Gentile, Leccino Mix Mix
Loreto Aprutino (PE) Citta` S. Angelo (PE) Citta` S. Angelo (PE) Loreto Aprutino (PE) Loreto Aprutino (PE) Loreto Aprutino (PE) Loreto Aprutino (PE) Loreto Aprutino (PE) Loreto Aprutino (PE) Farindola (PE) Rocca S. Giovanni (CH) Rocca S. Giovanni (CH) Rocca S. Giovanni (CH) Morro d’oro (TE) Cologna (TE) Ortona (CH) Casoli (CH) Casoli (CH) Orsogna (CH) Morro d’oro (TE) Crecchio (CH) Casoli (CH) Guardiagrele (CH) Loreto Aprutino (PE) Crecchio (CH) Crecchio (CH) Cappelle (PE) Orsogna (CH) Cepagatti (PE) Rocca S. Giovanni (CH) Orsogna (CH) Cologna (TE)
tech. systema % fly attack C C C C C P P P P P C C C D+C D+C C C C C C C C P C C P P C C C C C
2% 2.5% 2.5% 2.5% 4% 5% 5% 5% 5% 5% 5% 5% 5% 5% 7.5% 7.5% 7.5% 10% 10% 10% 15% 15% 25% 25% 30% 35% 35% 35% 45% 60% 60% 85%
a
P, pressure system; C, continuous system; D+C, destoner plus continuous system.
the water content with the volumetric titration of Karl Fisher) were from Riedel-deHae¨n (Seelze, Germany). All solvents used were analytical or HPLC grade (Merck & Co. Inc., Darmstadt, Germany). Samples. Thirty-two virgin olive oils produced from different industrial mills located in the Abruzzo region (Italy, December 2006) were analyzed. Samples differed in the percentage of fly attack, variety of olive cultivars, and technological system used (pressure or centrifugation, with or without a destoning phase) as reported in Table 1. The degree of infestation was calculated as the number of damaged olives per 100 fruits, considering both the presence of exit holes and grubs. Free Acidity and Peroxide Value (PV). These parameters were determined according to the official methods described in European Regulation EEC 2568/91 and amendments (27). PV was expressed as mequiv O2 kg-1 of oil. The samples were stored in the absence of light and at room temperature in order to measure PV after three months of storage. Relative standard deviation (RSD) of the free acidity method was 1.7, and RSD of the PV method was 1.4. Determination of Water Content in Virgin Olive Oil. The water content was analyzed with a TitroMatic 1S instrument (Crison Instruments, S.A.; Alella, Barcelona, Spain). This measurement uses a Karl Fischer titration based on a bivoltametric indication (2-electrode potentiometry). A solution of chloroform/Hydranal-solvent oil (a methanolic solvent) 2:1 (v/v) was used to dissolve the sample, and Hydranal-Titran 2 was used as a titrating reagent. Each sample was introduced three times, and the quantity of the sample was measured with the back weighting technique. The sample was dissolved in a solution of chloroform/Hydranal-solvent oil, and the titrating reagent was added until the equivalence point was reached. The quantity of water was expressed as mg of water/kg of oil (n ) 3). RSD of the water method was 3.5. Fatty Acid Composition. The fatty acid composition of oil samples was determined as methyl esters by capillary gas chromatography (GC)
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(Clarus 500 GC Perkin-Elmer Inc., Shelton, CT) analysis after alkaline treatment, according to Bendini et al. (28). Alkaline treatment was carried out by mixing 0.05 g of oil dissolved in 2 mL of n-hexane with 1 mL of 2 N potassium hydroxide in methanol according to Christie (29). Oxidation Stability Index (OSI) Time. These analyses were carried out in an eight-channel OSI instrument (Omnion, Decatur, IL, USA). Virgin olive oil samples (5.0 ( 0.1 g) were heated at 110 °C under atmospheric pressure, and air (150 mL min-1 of flow rate) was allowed to bubble through the oil. Under these conditions, the oxidative process reaches its final steps, and the short-chain volatile acids produced are recovered and measured conductimetrically in distilled water. The time required to produce a sudden increase in conductivity (due to volatile acid formation) determines an induction period (OSI time), expressed in hours and hundredths of hours, which can be used to measure the stability of oil. Extraction of Polar Phenolic Fraction. Phenolic compounds were extracted from virgin olive oil by a liquid-liquid extraction method according to Pirisi et al. (30). The dry extracts were dissolved in 0.5 mL of a methanol/water (50/50, v/v) solution and filtered through a 0.2 µm syringe filter (Whatman Inc., Clinton, NJ, USA). Extracts were frozen and stored at -43 °C. Electrophoretic Procedure. Capillary electrophoretic separation was performed by the capillary zone electrophoresis method proposed by Carrasco-Pancorbo et al. (31). A Beckman 5500 capillary electrophoresis instrument connected to a diode array detector was used. This method uses a capillary with 50 µm i.d. and a total length of 47 cm (40 cm to the detector) with a detection window of 100 × 200 µm, and a buffer solution containing 45 mM sodium tetraborate pH 9.3 Antioxidant Power (AOP) Determination. Phenolic extracts were measured in a FIA apparatus at a potential set at 0 mV vs Ag/AgCl. The apparatus consists of a Minipuls II peristaltic pump (Gilson, France), a high pressure injection valve model 7125 (Rheodine, USA) equipped with a 20 µL loop, an electrochemical cell model UniJet (BAS, West Lafayette, USA) mounted with a glassy carbon working electrode (3 mm diameter), and an amperometric detector AMEL 559 HPLC detector (AMEL, Milan, Italy) linked to a chart recorder (RC 102; Pharmacia, Sweden). The flow rate of phosphate buffer (pH 7.4) was 150 µL min-1. All extracts were injected in triplicate. The current produced during the electrochemical oxidation of the phenolic compounds was recorded. Quercetin was used as the reference compound, and the concentration of phenolic compounds was expressed as µg/ mL quercetin equivalent (QE); AOP was expressed as QE0, corresponding to QE. Statistical Analysis. Data were analyzed using Statistica 6.0 (Statsoft, Tulsa OK, USA) statistical software. The values reported are the averages of at least three repetitions (n ) 3), unless otherwise stated. Tukey’s honest significant difference (HSD) multiple comparison (oneway ANOVA) and Pearson’s linear correlations are both at p < 0.05. RESULTS AND DISCUSSION
Free Acidity and PV. The free acidity values of the oils studied ranged from 0.14% and 3.81%. Taking into account the acidity of the oil samples (32), there were 25 oils with very low acidity values (e0.8%) that could be classified as extra virgin olive oils; five samples (S22, S25, S26, S27, and S32) with an acidity between 0.8-2% that were defined as virgin olive oils, and finally two oils (S30 and S31) with an acidity higher than 2% that were considered as lampante olive oils. We demonstrated that the majority of oils with a fly attack more than 30% had an acidity higher than 0.8%, which means that these oils belonged to the category of virgin or lampante instead of extra virgin. Furthermore, the two samples that had suffered a fly attack higher than 50% had a very high acidity value and because of this belonged to the category of lampante olive oils. Regarding PV, the freshly pressed samples showed values from 5.3 to 19 mequiv O2 kg-1 oil and an average value of 9.90. These values are slightly higher than those usually obtained
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Go´mez-Caravaca et al. Table 3. Quantification Express as mg Analyte kg-1 Olive Oil of the Different Phenols by CE (mean ( SD, n ) 7) a
Table 2. Chemical Characteristics of Olive Oil Samplesa sample
FA
PV
PV 3
H2O
OSI
O/L
AOP
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32
0.5 0.3 0.2 0.3 0.4 0.5 0.6 0.6 0.7 0.4 0.3 0.4 0.6 0.7 0.2 0.4 0.3 0.1 0.6 0.3 0.6 0.9 0.3 0.8 1.0 0.9 1.2 0.7 0.5 3.8 2.3 1.9
8.4 6.6 5.9 9.9 9.3 10.2 8.8 8.4 10.9 7.9 11.3 7.9 12.2 7.1 8.8 9.7 6.1 5.3 13.8 7.8 9.8 9.1 7.5 11.2 12.1 11.3 19.0 8.3 10.4 14.8 11.9 14.9
10.0 9.1 9.2 8.2 9.9 11.0 10.0 13.2 10.2 11.1 9.0 12.0 13.0 10.0 7.2 10.7 8.2 6.4 11.0 10.7 16.8 15.0 8.2 12.0 15.5 12.2 20.2 10.0 14.8 17.4 17.4 15.2
1557 1121 904 1342 1585 1946 2053 1938 2095 2389 1407 1421 1479 927 951 974 1463 1002 1079 820 1365 1517 3332 1863 1366 2068 1573 1084 1257 1146 1176 1192
28.5 40.6 35.4 42.1 28.8 22.0 23.6 17.5 17.5 29.8 23.7 14.0 12.2 18.2 20.6 20.8 38.5 27.1 20.5 23.9 8.3 20.6 28.5 31.7 17.2 16.9 12.5 24.8 18.7 7.5 12.5 9.1
10.1 10.8 9.7 13.7 10.7 10.2 10.7 11.4 10.8 10.3 11.7 4.5 4.8 10.6 4.2 7.7 14.3 12.2 12.3 8.1 4.4 11.3 12.8 11.4 11.5 12.6 11.0 12.3 9.5 4.9 11.3 8.2
96.6 104.0 71.4 54.8 45.7 82.5 103.3 31.9 52.4 34.8 20.9 37.0 19.1 12.7 167.0 42.2 5.2 30.3 11.4 52.7 10.5 42.9 51.7 42.8 18.5 20.8 13.2 26.3 41.9 9.3 13.9 10.6
sample
simple phenolsb
lignansc
secoiridoidsc
total
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32
4.4 ( 0.6 1.9 ( 0.2 4.6 ( 0.2 4.7 ( 0.2 4.2 ( 0.4 5.1 ( 0.2 4.0 ( 0.3 2.4 ( 0.9 3.5 ( 0.3 4.6 ( 0.4 2.7 ( 0.1 3.4 ( 0.2 2.9 ( 0.2 2.3 ( 0.0 5.2 ( 0.2 6.9 ( 0.3 1.7 ( 0.1 3.7 ( 0.1 2.1 ( 0.1 3.4 ( 0.2 1.8 ( 0.6 1.8 ( 0.1 4.0 ( 0.3 5.4 ( 0.2 1.4 ( 0.5 1.1 ( 0.0 2.4 ( 0.8 2.6 ( 0.1 2.9 ( 0.1 1.5 ( 0.1 1.9 ( 0.1 1.0 ( 0.0
12.6 ( 3.2 11.9 ( 2.7 3.7 ( 0.5 8.9 ( 0.6 13.4 ( 3.8 11.7 ( 2.3 8.8 ( 2.0 10.8 ( 2.6 10.6 ( 2.00 6.2 ( 1.4 5.8 ( 0.4 27.5 ( 1.5 19.7 ( 1.2 7.8 ( 0.2 29.5 ( 1.5 19.5 ( 1.3 3.1 ( 0.4 2.6 ( 0.4 2.3 ( 0.3 6.5 ( 0.4 7.6 ( 1.6 10.3 ( 0.7 6.7 ( 0.8 9.6 ( 1.1 3.5 ( 0.3 2.6 ( 0.2 16.7 ( 3.3 3.3 ( 0.3 8.6 ( 1.3 3.8 ( 0.4 1.2 ( 0.3 3.7 ( 0.1
150.0 ( 7.2 264.5 ( 9.3 233.9 ( 11.4 193.8 ( 8.6 139.4 ( 9.9 98.3 ( 11.6 80.0 ( 10.5 74.8 ( 5.2 53.7 ( 9.2 141.0 ( 17.7 39.3 ( 2.3 63.7 ( 2.8 37.5 ( 0.2 24.4 ( 0.9 125.1 ( 2.8 116.8 ( 8.0 144.8 ( 9.7 77.6 ( 2.8 20.0 ( 0.5 102.6 ( 4.6 24.2 ( 1.1 115.1 ( 7.3 87.6 ( 8.9 158.4 ( 5.7 15.1 ( 1.0 18.0 ( 1.3 39.7 ( 3.4 40.7 ( 0.8 66.6 ( 4.4 20.0 ( 0.4 11.6 ( 0.7 14.2 ( 0.3
167.0 ( 4.9 278.4 ( 9.2 242.2 ( 11.9 207.5 ( 9.3 156.9 ( 9.8 115.1 ( 11.3 92.8 ( 11.9 88.0 ( 6.5 66.1 ( 9.8 151.8 ( 19.2 48.4 ( 3.1 98.1 ( 4.5 62.1 ( 1.4 34.6 ( 1.1 165.5 ( 3.7 144.8 ( 9.8 149.6 ( 10.1 83.9 ( 3.2 24.7 ( 0.9 112.5 ( 4.8 33.6 ( 1.4 127.2 ( 7.4 98.3 ( 10.0 173.4 ( 5.9 20.0 ( 1.7 21.7 ( 1.5 58.8 ( 3.2 47.2 ( 0.7 78.1 ( 4.1 25.3 ( 0.5 14.7 ( 0.9 18.9 ( 0.3
a
FA, free acidity percentage (g oleic acid on 100 g of oil); PV and PV3, peroxide values (mequiv O2 kg-1 oil) measured on fresh oils and after three months of oil storage; O/L, ratio between oleic and linoleic acids; H2O, water content (mg H2O kg-1 oil); OSI, oxidative stability index (hours); AOP, antioxidant power expressed as QE0 quercetin equivalent with potential set to 0 mV (µg quercetin mL-1 phenolic extract).
from fresh olive oils (11, 13). Another evaluation of PV was carried out after three months of storage because this period is long enough to observe the beginning of the oxidative reactions and to see differences in the PV. As shown in Table 2, after three months of storage the average of PV reached a mean of 11.70. The samples attacked to a higher degree were for the most part those that presented a stronger increase in the PV. As reported by other authors (12, 16–25) Bactrocera oleae attack has been positively correlated with both free acidity (r ) 0.77, p < 0.05) and PV (r ) 0.58, p < 0.05). These correlations were higher when the analyses were carried out after three months of oil storage: r ) 0.78 for acidity (data not shown) and r ) 0.63 for PV (in both cases for p < 0.05). In particular, when the percentage of infested olives was modest (
